Cell and tissue cultures are routinely utilized for commercial-scale production of various compounds including, for example, hormones, enzymes, proteins, antigens, food additives and natural pesticides.
Technology presently utilized for the production of cell and/or tissue culture at industrial scale is based on reusable glass or stainless steel bioreactor systems which are costly to set-up and maintain. Such bioreactor systems require cleaning and disinfecting between batches, and more intensive cleaning between product changeovers due to the need for expensive and time-consuming validation for cleanliness and presence of cleaning agent residue following cleaning.
In addition, these types of industrial bioreactor systems employ complicated and expensive mixing technologies such as impellers powered through expensive and complicated sterile seals; some expensive bioreactors comprise an airlift multipart construction, designed to provide mixing and gas saturation of the medium through bubbling of gas into the bioreactor. However, gas pressure, bubble size and the creation of undesirable shear forces in the medium necessitates the implementation of complicated aeration technologies. In addition, such bioreactors are designed according to the peak volume capacity that is required at the time. Thus, problems arise when scaling up from pilot plant bioreactor to large-scale bioreactor, or when the need arises to increase production beyond the capacity of existing bioreactors. The current alternative to operating a large-capacity bioreactor is to combine a number of smaller modular glass or stainless steel bioreactors whose total volume capacity matches requirements, while offering a degree of flexibility for increasing or reducing overall capacity. However, use of several smaller bioreactors increases cost and maintenance time and thus use of several small bioreactors is more expensive and labor intensive than the use of a single larger bioreactor.
Due to these limitations, culturing of plant cells in prior art bioreactors results in relatively expensive extractable products, including both secondary metabolites and recombinant proteins, which cannot compete commercially with comparable products produced by alternative production systems.
Presently, the only culture-based recombinant protein pharmaceutical produced in plant cell bioreactors is a commercial anti-viral vaccine for veterinary use in treatment of Newcastle virus. Other than this vaccine, however, there are presently only a very few secondary metabolite products produced by cell-culture in bioreactors, such as the plant metabolites paclitaxel (Taxol) and Shikonin.
At commercial scale, bioreactor systems traditionally employ permanent or semi-permanent growth chambers. Although disposable growth chambers are well known in the art, such growth chambers are typically utilized for small scale production volumes, such as in home brewing and for experimental laboratory work. Small scale bioreactors typically employ a disposable bag which can be utilized in laboratory settings.
Disposable bioreactors suitable for use with larger volumes have also been proposed. The requirements of agitation and aeration of the culture medium, which become more critical with scale-up of the reactor volume, are addressed in a number of ways in prior art systems. Applikon Biotechnology (The Netherlands) and Stedim Inc. (France) offer the Appliflex® single use bioreactor system using 50 liter flexible culture bags, which are designed for placement on a motorized platform which rocks the bag to provide aeration and agitation of the culture medium. A similar disposable bioreactor device is offered by Wave Biotech, LLD (Somerset, N.J.), which provides culture bags for volumes up to 1000 L, which are also aerated and agitated by a motorized platform. Hyclone Inc. (Logan, Utah), in conjunction with Baxter Biosciences, offers a disposable culture bag (Single Use Bioreactor “SUB”) designed for animal cell culture of up to 250 L, which is designed to retrofit stainless steel bioreactor vessels. Aeration and agitation is provided by a non-disposable impeller drive, which attaches to a complicated impeller unit integrated into the culture bag. US Patent Application No. 2005/0272146 to Hodge et al. discloses a 150 liter disposable bioreactor having impellor blades or other mechanical means for mixing. Yet another type of disposable bioreactor has a U-shaped bag, and requires a crane-like apparatus to agitate and aerate the culture medium through reciprocal lifting of the sides. Still another solution is based on a pressurized cuff surrounding the flexible culture bag, which is made to inflate and deflate at regular intervals, providing a squeezing type of mixing motion.
In all the abovementioned systems, support and aeration/agitation systems are complicated, costly, dedicated and limited in capacity. Thus, although the reactor vessel itself may be disposable and intended for single use, use of these systems requires costly tool-up and maintenance.
Disposable bioreactor devices using air for agitation and aeration of the culture have also been proposed, however, adaptation of air-bubble based aeration and mixing for large volumes is problematic. Many smaller volume bioreactors provide sufficient aeration with a single gas inlet and sparger, or other type of diffuser for the gas bubbles [see, for example, the Zeta bioreactor offered by Osmotec, Israel, (Agritech Israel, issue No. 1, Fall 1997, page 19)]. One disadvantage of such systems is that aeration performed by introducing very small air bubbles (from the diffuser) results in damage to cells, particularly in the case of plant cell cultures which are particularly sensitive to shear forces.
Proteins for pharmaceutical use have been traditionally produced in mammalian or bacterial expression systems. However, due to the relative simplicity of introducing genes into plants and plant cells for mass production of proteins and peptides, using, for example, plant molecular biology systems such as the Agrobacterium method, plant cell technology is becoming increasingly popular as an alternative protein expression system (Ma, J. K. C., Drake, P. M. W., and Christou, P. (2003) Nature reviews 4, 794-805).
Plant cell culture differs from bacterial or mammalian cell culture, not only in terms of metabolic requirements, but also as a function of the extreme fragility of the generally large sized plant cells to shear forces found in conventional industrial bioreactor. Thus, on the one hand, it is important to provide adequate mixing in the plant cell cultures, to ensure sufficient aeration of all aspects of the plant cell culture, but, on the other hand, this must be done in a manner suitable for the fragile plant cells grown in culture.
Thus, there is a constant need for improving on existing systems and devices for disposable cell/tissue culture, in order to provide greater yield and quality of the product, as well as improved cost-effectiveness. The present invention provides a high volume, disposable but reusable bioreactor, effective for use with a variety of cells/cell cultures for production of recombinant protein, in which the problems inherent in scale-up of the disposable reactor volume have been addressed.